Design and construction of diverse synthetic peptide and polypeptide libraries

ABSTRACT

The present invention concerns the design and construction of diverse peptide and polypeptide libraries. In particular, the invention concerns methods of analytical database design for creating datasets using multiple relevant parameters as filters, and methods for generating sequence diversity by directed multisyntheses oligonucleotide synthesis. The present methods enable the reduction of large complex annotated databases to simpler datasets of related sequences, based upon relevant single or multiple key parameters that can be individually directly defined. The methods further enable the creation of diverse libraries based on this approach, using multisynthetic collections of discrete and degenerate oligonucleotides to capture the diverse collection of sequences, or portions thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This is a non-provisional application claiming priority under 35 U.S.C.§119(e) from U.S. provisional patent application No. 60/849,035, filedOct. 2, 2006, the entire disclosure of which is expressly incorporatedby reference herein.

FIELD OF THE INVENTION

The present invention concerns the design and construction of diversepeptide and polypeptide libraries. In particular, the invention concernsmethods of analytical database design for creating datasets usingmultiple relevant parameters as filters, and methods for generatingsequence diversity by directed multisyntheses oligonucleotide synthesis.The present methods enable the reduction of large complex annotateddatabases to simpler datasets of related sequences, based upon relevantsingle or multiple key parameters that can be individually directlydefined. The methods further enable the creation of diverse librariesbased on this approach, using multisynthetic collections of discrete anddegenerate oligonucleotides to capture the diverse collection ofsequences, or portions thereof.

BACKGROUND OF THE INVENTION

The development of peptide- or polypeptide-based drug candidates oftenstarts with the screening of libraries of related peptide or polypeptidesequences. Thus the first step for the selection of therapeutic antibodycandidates usually is the creation of a highly diverse library ofantibody sequences.

Several methods for the design and construction of diverse antibodylibraries are known in the art.

It has been described that the diversity of a filamentous phage-basedcombinatorial antibody library can be increased by shuffling of theheavy and light chain genes (Kang et al., Proc. Natl. Acad. Sci. USA,88:11120-11123, (1991)) or by introducing random mutations into thelibrary by error-prone polymerase chain reactions (PCR) (Gram et al.,Proc. Natl. Acad. Sci. USA, 89:3576-3580, (1992)). The use of definedframeworks as the basis for generating antibody libraries has beendescribed by Barbas et al., Proc. Natl. Acad. Sci. USA 89:4457-4461(1992) (randomizing CD3-H3); Barbas et al., Gene 137:57-62 (2003)(extending randomization to V_(κ) CDR3); and Hayanashi et al.,Biotechniques 17:310 (1994) (simultaneous mutagenesis of antibody CDRregions by overlap extension and PCR). Others report combination ofCDR-H3 libraries with a single V_(L) gene (Nissim et al., EMBO J.13:692-698 (1994)), a limited set of V_(L) genes (De Kruif et al., J.Mol. Biol. 248:97-105 (1995)); or a randomized repertoire of V_(L) genes(Griffiths et al., EMBO J. 13:3245-3260 (1994)).

See also U.S. Pat. Nos. 5,667,988; 6,096,551; 7,067,284 describingmethods for producing antibody libraries using universal or randomizedimmunoglobulin light chains.

Knappik et al., J. Mol. Biol. 296:57-86 (2000) describe a differentconcept for designing and constructing human antibody libraries,designated HuCAL (Human Combinatorial Antibody Libraries). This approachis based on the finding that each of the human V_(H) and V_(L)subfamilies that is frequently used during an immune response isrepresented by one consensus framework, resulting in seven HuCALconsensus genes for heavy chains and seven HuCAL consensus genes forlight chains, which yield 49 possible combinations. All genes are madeby total synthesis, taking into consideration codon usage, unfavorableresidues that promote protein aggregation, and unique and generalrestriction sites flanking all CDRs. The approach leads to thegeneration of modular antibody genes containing CDRs that can beconverted into different antibody formats, as needed. The design andsynthesis of HuCAL antibody libraries is described in U.S. Pat. Nos.6,300,064; 6,696,248; 6,706,484; and 6,828,422.

Despite these and other advances there a great need for new, efficientmethods for the design and construction of highly diverse (poly)peptide,such as antibody, libraries.

SUMMARY OF THE INVENTION

The present invention concerns the design and construction of diversepeptide and polypeptide libraries.

In one aspect, the invention concerns a method for diversity analysis ofa database comprising related amino acid sequences characterized by atleast one shared sequence motif, comprising the steps of:

(a) aligning the related amino acid sequences;

(b) creating a first dataset by applying a predetermined combination oftwo or more filters to the related amino acid sequences comprising theshared sequence motif;

(c) analyzing the first dataset for positional amino acid usagefrequency within the shared sequence motif; and

(d) creating a second dataset characterized by a minimum threshold aminoacid usage frequency at one or more amino acid positions within theshared sequence motif.

In step (d) a minimum threshold amino acid usage frequency can beassigned to any and all amino acid positions within the shared sequencemotif.

In one particular embodiment, a minimum threshold amino acid usagefrequency is assigned to the majority of amino acid positions within theshared sequence motif. In another particular embodiment, a minimumthreshold amino acid usage frequency is assigned to all amino acidpositions within the shared sequence motif. In various embodiments, theminimum threshold amino acid usage frequencies assigned to specificamino acid positions within the shared sequence motif can be identicalor different.

In a further embodiment, the minimum threshold amino acid usagefrequency is set to provide a minimum sum amino acid usage for themajority of amino acid positions within the shared sequence motif.

In a still further embodiment, the minimum threshold amino acid usagefrequency is set to provide a minimum sum amino acid usage for all aminoacid positions within said shared sequence motif.

The minimum sum amino acid usage can be set to any desired level, and inparticular embodiments it is at least about 40%, or at least about 45%,or at least about 50%, or at least about 55%, or at least about 60%, orat least about 65%, or at least about 70%, or at least about 75%, or atleast about 80%, or at least about 85%, or at least about 90%.

In another embodiment, the related amino acid sequences are antibodysequences.

In yet another embodiment, the related amino acid sequences compriseantibody heavy chain sequences.

In a further embodiment, the related amino acid sequences compriseantibody light chain sequences.

If the related amino acid sequences are antibody sequences, the sharedsequence motif may, for example, be a CDR sequence, such as a CDR1, CDR2or CDR3 sequence.

There are no limitations on the nature or number of the filters that canbe used in step (b) of the method of the present invention. In aparticular embodiment, in the case of antibody sequences, thepredetermined combination of filters can be selected from the groupconsisting of (1) the isotype of an antibody heavy or light chain; (2)the length of one or more of CDR1, CDR2 and CDR3 sequences; (3) thepresence of one or more predetermined amino acid residues at one or morepredetermined positions within one or more CDR1, CDR2 and CDR3sequences; (4) type of framework; (5) antigen to which the antibodybinds; (6) affinity of the antibody; and (7) positional amino acidresidues outside the CDR sequences.

In a further embodiment, at least one of the antibody heavy and/or lightchain CDR1, CDR2 and CDR3 sequences is size matched. This parameter can,for example, be combined with the isotype of the antibody heavy and/orlight chain sequences, as an additional filter.

In various embodiments, the positional amino acid usage frequency is atleast about 3%, or at least about 5%, or at least about 10%, or at leastabout 15%, or set between about 3% and about 15%, or between about 5%and about 10%.

In another embodiment of the methods of the present invention, the samepositional amino acid usage frequency characterizes each amino acidwithin said CDR sequence. In an alternative embodiment, the positionalamino acid usage frequencies differ at at least two amino acid residueswithin said CDR sequence.

In another embodiment, the predetermined combination of filters includesthe type of framework.

In yet another embodiment, both antibody heavy and light chain sequencesare analyzed. Optionally, the antibody heavy chain sequences are pairedto predetermined antibody light chain characteristics, or the antibodylight chain sequences are paired to predetermined antibody heavy chaincharacteristics.

In a further embodiment, the related antibody sequences are from atleast one functional antibody.

In a still further embodiment, at least one of the filters applied instep (b) of the method of the invention is the germline sequence mostsimilar to the framework sequence of the heavy and/or light chain of afunctional antibody.

Without limitation, the functional antibody may, for example, bind to apolypeptide selected from the group consisting of cell surface andsoluble receptors, cytokines, growth factors, enzymes; proteases; andhormones. Thus, the antibody may bind to a cytokine, such as aninterleukin, e.g. IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-11,IL-12, IL-15, IL-17, IL-18, IL-23, and their respective family members.Alternatively, the cytokine may, for example, be selected from the groupconsisting of interferons-α, -β, and -γ (IFN-α, -β, and -γ), tumornecrosis factor-α, and -β (TNF-α and -β), TWEAK, RANKL, BLys, RANTES,MCP-1, MIP-1α, MIP-1β, SDF-1, colony stimulating factor (CSF),granulocyte colony stimulating factor (G-CSF), and granulocytemacrophage colony stimulating factor (GMCSF).

The polypeptide to which the antibody binds may also be a growth factor,including, without limitation, nerve growth factor (NGF), insulin-likegrowth factor 1 (IGF-1), epidermal growth factor (EGF), plateled derivedgrowth factor (PDGF), vascular endothelial growth factor (VEGF),placental growth factor (PLGF), tissue growth factor-α (TGF-α), andtissue growth factor-β (TGF-β).

In another embodiment, the functional antibody binds to a hapten, e.g.Dig, Bio, DNP, or FITC.

In yet another embodiment of the methods herein, the related amino acidsequences originate from members of a family of secreted orextracellular proteins, which can be cytokines, for example.

In a specific embodiment, the cytokine is interferon-α, and the relatedamino acid sequences are sequences of IFN-α subtypes.

In a particular embodiment, the invention further comprises the step ofsynthesizing a physical library of related amino acid sequences that isdesigned with the aid of the datasets identified.

In a certain embodiment of this method, the library is synthesized bygenerating a discrete number of defined or degenerate oligonucleotidessuch that only defined amino acids are generated.

In a further embodiment, the diversity of the physical library producedexceeds the diversity of a library which is a physical representation ofthe datasets identified. This can, for example, result from the factthat at least one amino acid not meeting the minimum threshold aminoacid usage frequency is also synthesized to provide said diversity.

In a still further embodiment, the diversity of the physical libraryproduced is less than the diversity of a library which is a physicalrepresentation of the datasets identified. This can results from thefact that not all amino acids meeting the minimum threshold amino acidusage frequency are synthesized, for example.

In another embodiment, the dataset comprises antibody heavy and/or lightchain sequence, which may include one or more CDRs.

In yet another embodiment, the CDRs are cloned into a scaffold offramework sequences, which may, optionally, be the most frequently usedframework sequences in the database comprising said CDRs.

The physical library may be expressed using any expression system,including all prokaryotic and eukaryotic expression systems.

In a specific embodiment, the physical library is expressed anddisplayed using a phagemid display, mRNA display, microbial celldisplay, mammalian cell display, microbead display technique, antibodyarray, or display based on protein-DNA linkage.

In another embodiment of the invention, the library is screened for oneor more chemical and/or biological properties of its members. Suchproperties may include, without limitation, half-life, potency,efficacy, binding affinity, and immunogenicity.

In yet another embodiment, amino acid side-chain diversity is introducedinto members of the library at one or more amino acid positions.

In a particular embodiment, the amino acid side-chain diversity isintroduced by providing amino acid residues with at least two differentside-chain chemical functionalities at said amino acid position orpositions.

In other embodiments, at least 30%, or at least 50%, or at least 55%, orat least 60% of all amino acid chemistries are represented at each aminoacid position.

Preferably, amino acid said side-chain diversity is introduced by usingcombinatorial degenerate oligonucleotide synthesis.

In another aspect, the invention concerns a method of producing acombinatorial library of peptide or polypeptide sequences, comprisingintroducing amino acid side-chain chemical diversity into the peptide orpolypeptide sequences at two or more amino acid positions, usingcombinatorial oligonucleotide synthesis.

In one embodiment, the amino acid side-chain chemical diversity isdesigned to mimic naturally occurring diversity in said peptide orpolypeptide sequences.

The library can be any type of library, including, but not limited to,antibody libraries.

In a specific embodiment, the antibody library comprises antibody heavychain variable domain sequences.

In another embodiment, the library comprises antibody light chainvariable domain sequences.

In yet another embodiment, the library is a combinatorial single-chainvariable fragment (scFv) library.

In a further embodiment, the antibody library is a library of Fab, Fab′,or F(ab′)₂ fragments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a summary of the representative steps of the design andconstruction of a diverse human antibody library.

FIG. 2: Frequency analysis of V_(κ) CDR1, 2, and 3; determination ofabsolute usage by position.

FIG. 3: V_(κ) I light chain threshold analysis. No individual aminoacids are reported below 10% usage.

FIG. 4: V_(κ) I light chain threshold analysis. No individual aminoacids are reported below 5% usage.

FIG. 5: Synthesizing light chain CDR1 diversity.

FIG. 6: V_(H)3 heavy chain synthetic library threshold analysis; length10 residues. The threshold percentage usage has been individually setfor each amino acid position, and is between 3% and 10%.

FIG. 7: Oligonucleotides used to synthesize the library designed asshown in FIG. 6.

FIG. 8: Determination of germline origin of productive anti-TNF-αantibody heavy chain.

FIG. 9: Dendrogram alignment illustrating the germline origin ofproductive anti-TNF-α antibody heavy chain.

FIG. 10: Determination of germline origin of productive anti-TNF-αantibody light chain.

FIG. 11: Dendrogram alignment illustrating the germline origin ofproductive anti-TNF-α antibody light chain.

FIG. 12: V_(κ)1 light chain synthetic library diversity.

FIG. 13: Frequency analysis of V_(H)3 CDR1 and CDR2.

FIG. 14: CDR1 and CDR2 threshold analysis—part one.

FIG. 15: CDR1 an CDR2 threshold analysis—part two.

FIG. 16: V_(H)3 heavy chain synthetic library diversity.

FIG. 17: Design of V_(H)3 heavy chain synthetic library diversity basedon anti-digoxigenin antibody D2E7.

FIG. 18: Anti-digoxigenin antibody Ig λ light chain variable region andheavy chain variable region sequences.

FIG. 19: Determination of germline origin of anti-digoxigenin antibodyheavy and light chains.

FIG. 20: Hapten analysis for λ length matched CO framework.

FIG. 21: Hapten analysis for H3—length 8 amino acids.

FIG. 22: Alignment of amino acid residues 32-38 of IFN-α subtypes.

FIG. 23: Oligonucleotide design to encode desired IFN-α diversity.

FIG. 24: Amino acids categorized by side-chain chemistries.

FIG. 25: Encoding chemically probed diversity positions.

FIG. 26: CDR3 containing chemically probed diversity.

FIG. 27: Encoding CDR3 heavy chain diversity with chemical probe sets.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994), provides one skilled in the art with a general guide to manyof the terms used in the present application.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

The phrase “shared sequence motif” is used herein in the broadest senseand is used to refer to a pattern of amino acid residues common betweentwo or more peptide or polypeptide sequences. Sequence motifs can bereadily identified by a variety of pattern discovery algorithms, such asthose discussed in the detailed description of the invention.

In the context of the present invention, the term “antibody” (Ab) isused in the broadest sense and includes immunogloblins which exhibitbinding specificity to a specific antigen as well as immunoglobulins andother antibody-like molecules which lack antigen specificity.Polypeptides of the latter kind are, for example, produced at low levelsby the lymph system and, at increased levels, by myelomas. In thepresent application, the term “antibody” specifically covers, withoutlimitation, monoclonal antibodies, polyclonal antibodies, and antibodyfragments.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby covalent disulfide bond(s), while the number of disulfide linkagesvaries between the heavy chains of different immunoglobulin isotypes.Each heavy and light chain also has regularly spaced intrachaindisulfide bridges. Each heavy chain has, at one end, a variable domain(V_(H)) followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light- andheavy-chain variable domains, Chothia et al, J. Mol. Biol. 186:651(1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA. 82:4592 (1985).

The term “variable” with reference to antibody chains is used to referto portions of the antibody chains which differ extensively in sequenceamong antibodies and participate in the binding and specificity of eachparticular antibody for its particular antigen. Such variability isconcentrated in three segments called hypervariable regions both in thelight chain and the heavy chain variable domains. The more highlyconserved portions of variable domains are called the framework region(FR). The variable domains of native heavy and light chains eachcomprise four FRs (FR1, FR2, FR3 and FR4, respectively), largelyadopting a n-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the β-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991), pages 647-669). Theconstant domains are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody in antibody-dependent cellular toxicity.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e., residues 30-36(L1), 46-55 (L2) and 86-96 (L3) in the light chain variable domain and30-35 (H1), 47-58 (H2) and 93-101 (H3) in the heavy chain variabledomain; MacCallum et al., J Mol Biol. 1996.

The term “framework region” refers to the art recognized portions of anantibody variable region that exist between the more divergent CDRregions. Such framework regions are typically referred to as frameworks1 through 4 (FR1, FR2, FR3, and FR4) and provide a scaffold for holding,in three-dimensional space, the three CDRs found in a heavy or lightchain antibody variable region, such that the CDRs can form anantigen-binding surface.

Depending on the amino acid sequence of the constant domain of theirheavy chains, antibodies can be assigned to different classes. There arefive major classes of antibodies IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

The heavy-chain constant domains that correspond to the differentclasses of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable domain thereof. Examples ofantibody fragments include, but are not limited to, Fab, Fab′, F(ab′)₂,Dab, and Fv fragments, linear antibodies, single-chain antibodymolecules, diabodies, and multispecific antibodies formed from antibodyfragments.

The term “monoclonal antibody” is used to refer to an antibody moleculesynthesized by a single clone of B cells. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. Thus, monoclonal antibodies may be made by the hybridoma methodfirst described by Kohler and Milstein, Nature 256:495 (1975); Eur. J.Immunol. 6:511 (1976), by recombinant DNA techniques, or may also beisolated from phage antibody libraries.

The term “polyclonal antibody” is used to refer to a population ofantibody molecules synthesized by a population of B cells.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994). Single-chain antibodies are disclosed, for example in WO88/06630 and WO 92/01047.

As used herein the term “antibody binding regions” refers to one or moreportions of an immunoglobulin or antibody variable region capable ofbinding an antigen(s). Typically, the antibody binding region is, forexample, an antibody light chain (VL) (or variable region thereof), anantibody heavy chain (VH) (or variable region thereof), a heavy chain Fdregion, a combined antibody light and heavy chain (or variable regionthereof) such as a Fab, F(ab′)₂, single domain, or single chain antibody(scFv), or a full length antibody, for example, an IgG (e.g., an IgG1,IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

The term “threshold frequency of occurrence” refers to a criterion ofthe invention which requires that a selected sequence for use in alibrary herein be derived from a sequence which has been determined tobe a sequence favored to be expressed. Depending on the ultimate goal,such as the required degree of diversity, the desired size of library,the “threshold frequency of occurrence” can be set at different levels.

The term “amino acid” or “amino acid residue” typically refers to anamino acid having its art recognized definition such as an amino acidselected from the group consisting of: alanine (Ala); arginine (Arg);asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gin);glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile):leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe);proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine(Tyr); and valine (Val) although modified, synthetic, or rare aminoacids may be used as desired. Thus, modified and unusual amino acidslisted in 37 CFR 1.822(b)(4) are specifically included within thisdefinition and expressly incorporated herein by reference. Amino acidscan be subdivided into various sub-groups. Thus, amino acids can begrouped as having a nonpolar side chain (e.g., Ala, Cys, Ile, Leu, Met,Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); apositively charged side chain (e.g., Arg, His, Lys); or an unchargedpolar side chain (e.g., Asn, Cys, Gin, Gly, His, Met, Phe, Ser, Thr,Trp, and Tyr). Amino acids can also be grouped as small amino acids(Gly, Ala), nucleophilic amino acids (Ser, His, Thr, Cys), hydrophobicamino acids (Val, Leu, Ile, Met, Pro), aromatic amino acids (Phe, Tyr,Trp, Asp, Glu), amides (Asp, Glu), and basic amino acids (Lys, Arg)(see, FIG. 25).

The term “conserved amino acid residue” refers to an amino acid residuedetermined to occur with a high frequency, typically at least 50% ormore (e.g., at about 60%, 70%, 80%, 90%, 95%, or higher), for a givenresidue position in two or more amino acid sequences compared.

The term “semi-conserved amino acid residue” refers to amino acidresidues determined to occur with a high frequency between two or moreamino acid sequences compared for a given residue position. When 2-3residues, preferably 2 residues, that together, are represented at afrequency of about 40% of the time or higher (e.g., 50%, 60%, 70%, 80%,90% or higher), the residues are determined to be semi-conserved.

The term “variable amino acid residue” refers to amino acid residuesdetermined to occur with a variable frequency between two or moresequences compared for a given residue position. When many residuesappear at a given position, the residue position is determined to bevariable.

The term “variability profile” refers to the cataloguing of amino acidsand their respective frequencies of occurrence present at a particularamino acid position within a polypeptide sequence, such as within a CDRof an antibody.

The term “polynucleotide(s)” refers to nucleic acids such as DNAmolecules and RNA molecules and analogs thereof (e.g., DNA or RNAgenerated using nucleotide analogs or using nucleic acid chemistry). Asdesired, the polynucleotides may be made synthetically, e.g., usingart-recognized nucleic acid chemistry or enzymatically using, e.g., apolymerase, and, if desired, be modified. Typical modifications includemethylation, biotinylation, and other art-known modifications. Inaddition, the nucleic acid molecule can be single-stranded ordouble-stranded and, where desired, linked to a detectable moiety.

The term “mutagenesis” refers to, unless otherwise specified, any artrecognized technique for altering a polynucleotide or polypeptidesequence. Preferred types of mutagenesis include error prone PCRmutagenesis, saturation mutagenesis, or other site directed mutagenesis.

The term “vector” is used to refer to a rDNA molecule capable ofautonomous replication in a cell and to which a DNA segment, e.g., geneor polynucleotide, can be operatively linked so as to bring aboutreplication of the attached segment. Vectors capable of directing theexpression of genes encoding for one or more polypeptides are referredto herein as “expression vectors.”

The term “primer,” as used herein, refers to a polynucleotide whetherpurified from a nucleic acid restriction digestion reaction or producedsynthetically, which is capable of acting as a point of initiation ofnucleic acid synthesis when placed under conditions in which synthesisof a primer extension product which is complementary to a nucleic acidstrand is induced. Such conditions may include the presence ofnucleotides and a DNA polymerase, reverse transcriptase and the like, ata suitable temperature and pH. The primer is preferably single stranded,but may also be in a double stranded form. The primer must besufficiently long to prime the synthesis of extension products in thepresence of the agents for polymerization. The exact lengths of theprimers will depend on many factors, including the complexity of thetarget sequence, temperature and the source of primer. A primertypically contains about 15 to about 25 nucleotides, but shorter andlonger primers may also be used. Shorter primers generally requirecooler temperatures to form stable complexes with the template.

A “phage display library” is a protein expression library that expressesa collection of cloned protein sequences as fusions with a phage coatprotein. Thus, the phrase “phage display library” refers herein to acollection of phage (e.g., filamentous phage) wherein the phage expressan external (typically heterologous) protein. The external protein isfree to interact with (bind to) other moieties with which the phage arecontacted. Each phage displaying an external protein is a “member” ofthe phage display library.

An “antibody phage display library” refers to a phage display librarythat displays antibodies or antibody fragments. The antibody libraryincludes the population of phage or a collection of vectors encodingsuch a population of phage, or cell(s) harboring such a collection ofphage or vectors. The library can be monovalent, displaying on averageone single-chain antibody or antibody fragment per phage particle ormulti-valent displaying, on average, two or more antibodies or antibodyfragments per viral particle. The term “antibody fragment” includes,without limitation, single-chain Fv (scFv) fragments and Fab fragments.Preferred antibody libraries comprise on average more than 10⁶, or morethan 10⁷, or more than 10⁸, or more than 10⁹ different members.

The term “filamentous phage” refers to a viral particle capable ofdisplaying a heterogenous polypeptide on its surface, and includes,without limitation, fl, fd, Pfl, and M13. The filamentous phage maycontain a selectable marker such as tetracycline (e.g., “fd-tet”).Various filamentous phage display systems are well known to those ofskill in the art (see, e.g., Zacher et al. Gene 9: 127-140 (1980), Smithet al. Science 228: 1315-1317 (1985); and Parmley and Smith Gene 73:305-318 (1988)).

The term “panning” is used to refer to the multiple rounds of screeningprocess in identification and isolation of phages carrying compounds,such as antibodies, with high affinity and specificity to a target.

B. Detailed Description

Techniques for performing the methods of the present invention are wellknown in the art and described in standard laboratory textbooks,including, for example, Ausubel et al., Current Protocols of MolecularBiology, John Wiley and Sons (1997); Molecular Cloning: A LaboratoryManual, Third Edition, J. Sambrook and D. W. Russell, eds., Cold SpringHarbor, N.Y., USA, Cold Spring Harbor Laboratory Press, 2001; O'Brian etal., Antibody Phage Display, Methods and Protocols, Humana Press, 2001;Phage Display: A Laboratory Manual, C. F. Barbas III et al. eds., ColdSpring Harbor, N.Y., USA, Cold Spring Harbor Laboratory Press, 2001; andAntibodies, G. Subramanian, ed., Kluwer Academic, 2004. Mutagenesis can,for example, be performed using site-directed mutagenesis (Kunkel etal., Proc. Natl. Acad. Sci USA 82:488-492 (1985)). PCR amplificationmethods are described in U.S. Pat. Nos. 4,683,192, 4,683,202, 4,800,159,and 4,965,188, and in several textbooks including “PCR Technology:Principles and Applications for DNA Amplification”, H. Erlich, ed.,Stockton Press, New York (1989); and “PCR Protocols: A Guide to Methodsand Applications”, Innis et al., eds., Academic Press, San Diego, Calif.(1990).

Information concerning antibody sequence analysis using the Kabatdatabase and Kabat conventions may be found, e.g., in Johnson et al.,The Kabat database and a bioinformatics example, Methods Mol Biol. 2004;248:11-25; and Johnson et al., Preferred CDRH3 lengths for antibodieswith defined specificities, Int Immunol. 1998, December; 10(12):1801-5.

Information regarding antibody sequence analysis using Chothiaconventions may be found, e.g., in Chothia et al., Structuraldeterminants in the sequences of immunoglobulin variable domain, J MolBiol. 1998 May 1; 278(2):457-79; Morea et al., Antibody structure,prediction and redesign, Biophys Chem. 1997; 68(1-3):9-16.; Morea etal., Conformations of the third hypervariable region in the VH domain ofimmunoglobulins; J Mol Biol. 1998, 275(2):269-94; Al-Lazikani et al.,Standard conformations for the canonical structures of immunoglobulins,J Mol Biol. 1997, 273(4):927-48. Barre et al., Structural conservationof hypervariable regions in immunoglobulins evolution, Nat Struct Biol.1994, 1(12):915-20; Chothia et al., Structural repertoire of the humanVH segments, J Mol Biol. 1992, 227(3):799-817 Conformations ofimmunoglobulin hypervariable regions, Nature. 1989, 342(6252):877-83;and Chothia et al., Review Canonical structures for the hypervariableregions of immunoglobulins, J Mol Biol. 1987, 196(4):901-17).

1. In Silico Design of Diverse (Poly)Peptide Libraries

According to the present invention, the design of diverse (poly)peptidelibraries starts with the use of a database of related (poly)peptidesequences of interest, and, typically, the identification of sequencemotifs that are shared by individual members of the library. Variouscomputer programs for identifying sequence motifs in polypeptides arewell known in the art and can be used on-line. Thus, for example,sequence motifs can be identified using the ELPH (a general-purposeGibbs sampler for finding motifs in a set of DNA or protein sequences),MEME (Multiple EM for Motif Elicitation system that allows one todiscover motifs of highly conserved regions in groups of related DNA orprotein sequences); PPSEARCH (allows to search sequences for motifs orfunctional patterns in the PROSITE database (EBI)); emotif (a researchsystem that forms motifs for subsets of aligned sequences, and ranks themotifs that it finds by both their specificity and the number ofsupplied sequences that it covers (Stanford Bioinformatics Group)); andthe like.

In the next step, one or more sequence motifs identified are aligned toeach other, and subdivided into separate datasets, each dataset beingcharacterized by sharing a predetermined combination of parameterscharacteristic of one or more of the aligned sequence motifs. Suchparameter can, for example, be the length, the subfamily in which aparticular sequence motif belongs, the species from which the sequencederives, biological function, etc. The datasets characterized by a givencombination of two or more parameters are then analyzed by position foramino acid frequency usage to identify key amino acid usage inindividual stretches of amino acids within the datasets.

Alignment of the sequence motifs can be achieved in various ways thatare within the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

Determination of amino acid frequency usage can be based on theappearance of high degree (typically at least 50%), and preferablycomplete, identity in all members of the dataset in a given position(conserved amino acid residue), or appearance of an amino acid residuein two or more members (preferably the majority) of the dataset for agiven residue position. Additional datasets characterized by one or moreadditional parameters can then be created, not all of which need to besequence related.

For example, if the goal is to design a diverse antibody library,antibody heavy and light chain CDR sequences present in the Kabatdatabase, an electronic database containing non-redundant rearrangedantibody sequences, can be analyzed for positional frequencies withunique combinations (filters) of predetermined parameters. The Kabatdatabase contains antibody protein sequences, which are annotated uponsubmission. Information from the Kabat database can be imported intoother environments, such as, for example, a Microsoft Access database,which allows for convenient application of filters, and the results canbe tabulated and further analyzed in using any other software,including, for example, Excel.

The approach of the present invention allows for simultaneous filteringof both antibody heavy and light chain sequences, using a wide array ofparameters (filters) and combination of parameters (filters). Thus, thegeneration of diversity datasets for particular heavy chains can belinked to light chain restrictions of choice. For example, filters forthe analysis of antibody heavy chain CDR sequences may include one ormore of (1) pairing with a certain light chain type (e.g. kappa (κ) orlambda (λ)); (2) CDR size (e.g. CDR1=6 residues; CDR2=13 residues); and(3) CDR3 subfamily (e.g. V_(H)1 vs. V_(H)3). In the light chains, allCDRs may be size matched. For example, it can be pre-determined thatCDR1=7, CDR2=10, and CDR3=8 amino acid residues. In addition, or in thealternative, the light chains can be filtered (sub-divided) based on thetype of the light chain subfamily (e.g. κ1 or κ3 subfamily).

Thus, for example, heavy chain diversity analysis can be performed basedupon pairing with κ light chains, but the analysis may also be furtherrestricted to those heavy chain sequences that pair with V_(κ)3subfamily light chains, or to κ light chains bearing a CDR3 containing alength of 8 amino acids, or a combination of both.

Additional filters for covariant analysis of antibody heavy and/or lightchains may include, without limitation, isotype, antigen type, affinity,and/or positional residues not related to CDR or the type or subtype ofantibody chain.

In addition, the present invention enables the design of themedlibraries, based upon “productive” heavy and light chain pairings. Thusvarious antibodies to the same antigen, including commercial antibodies,can be subjected to diversity analysis to identify antibodies that aremost likely to succeed in human therapy.

If the goal is the design of themed antibody libraries, based uponproductive heavy and light chain pairings, one or more productive (e.g.commercial) antibodies are selected to a selected antigen. Next, thegermline origins to both the heavy and light chains are determined, andthe heavy and light chain CDR sequences of the same type (e.g. V_(H)3,V_(κ)1) are subjected to the type of multivariate analysis describedabove to create diversity datasets. Preferably, the analysis should bebased only on size-matched CDRs.

In the methods of the present invention, alignment and application offilters are followed by positional analysis in order to determine thepositional frequency of individual amino acids or groups of amino acidswithin the previously created datasets, and to generate diversitydatasets, such as CDR diversity datasets. After determining the absolutepositional amino acid usage for each amino acid position of interest,the thresholds for percentage usage and for sum usage of amino acids canbe lowered, in order to accommodate greater coverage of diversity. Thus,for example, the required total coverage may be set to more than 80%,with no individual amino acid being represented below 10%.

The in silico modeling can be continually updated with additionalmodeling information, from any relevant source, e.g., from gene andprotein sequence and three-dimensional databases and/or results frompreviously tested polypeptides, such as antibodies, so that the insilico database becomes more precise in its predictive ability.

In addition, the in silico subsets can be supplemented with results ofbiological assays, such as binding affinity/avidity results, biologicalactivity of previously tested antibodies. In this way, structuralfeatures can be more closely correlated with expected performance for anintended use.

Design of CDR diversity datasets is followed by the synthesis of acollection of combinatorial (degenerate) oligonucleotide sequencesproviding the required diversity, and cloning of the collection on thebackground of a suitable template.

2. Construction of Diverse (Poly)Peptide Libraries

After the creation of combinatorial positional diversity data sets asdescribed above, physical combinatorial diversity sets can be generatedby multisyntheses oligonucleotide synthesis. According to the presentinvention, instead of using a mutagenic code or mixed codon trimers,discrete degenerate oligonucleotide collections are generated that canbe quantitatively restricted or relaxed to physically represent thecombinatorial diversity sets produced through the foregoing analysis anddesign. Relaxing the criteria enables capture of the desired diversitythrough synthesis of fewer oligonucleotide probes, or to rationallyexpand the diversity set if the ability to clone the collection exceedsthe predicted collection generated through diversity analysis. Inaddition, the physical combinatorial diversity sets can includeside-products not found in the virtual diversity sets, with or withoutadditional rule sets. This approach is most helpful in the field ofcombinatorial antibody library generation, but can also be rationallyextended into other appropriate applications, such as to generatinglibraries of various polypeptide classes (e.g. growth factor libraries),etc. It is important to note that the physical library does notnecessarily need to contain members comprising all amino acids at anygiven position that were identified by setting the threshold percentageusage as described above. For various reasons, for example in order toreduce the number of oligonucleotides needed, it may be advantageous toomit certain amino acid(s) at a given position. Alternatively or inaddition, it is possible to increase coverage and diversity of thelibrary by synthesizing members with amino acid residue(s) at a givenposition that did not meet the pre-set threshold frequency usage. Thetwo approaches can be combined, i.e. certain amino acid residues presentin the in silico diversity data sets may be omitted while others, notrepresented in the in silico diversity data sets at a given position maybe added.

The first step in the creation of peptide or polypeptide librariesherein is the reverse translation of a collection of amino acids formultiplexed synthesis to contain an entire positional collection.Reverse translation tools are well known in the art and are commerciallyavailable. For example, the Java-based backtranslation tool ofEntelechon (DE) translated proteins into nucleotides sequences withadapted codon usage, and allows optimization of a sequence forexpression in specific organisms. In a preferred embodiment, the methodsof the present invention employ an automated reverse translationalgorithm capable of synthesizing discrete and degenerate sets ofoligonucleotides to represent the diversity tables created by in silicoanalysis. This algorithm can include or exclude particular codons andeven incorporate non-equimolar degeneracies to more accurately achievenot only the diversity of the dataset but also the relativedistributions.

The number of oligonucleotides needed can be restricted by selectingdegenerate bases to simultaneously encoding more than one of thefrequently used amino acids at a time. In addition, such degeneratebases can be restricted to avoid rare codons of the species of interest.For example, if the collection is synthesized in E. coli, the use ofrare arginine codon usage for E. coli can be restricted in the reversetranslation. In addition, it is known that not all amino acids are usedwith the same frequency. Therefore, non-equimolar mixes can be used tomore accurately reflect the profile of the virtual (in silico) diversitytables.

Where positional diversity requires the synthesis of moreoligonucleotides than desired, diversity can be arbitrarily defined witha chemical probe collection. Thus, amino acid side-chain chemistries canbe captured within subsets of amino acids, such as small, hydrophobic,aromatic, basic, acidic, amide, nucleophilic, etc. amino acids canconstitute such subsets. As the Examples will illustrate, suchchemically probed diversity positions can be synthesized by using a muchsmaller number of oligonucleotides than would otherwise be required.Chemically probed diversity covers much of the naturally occurringdiversity, and provides broad interactive chemistries.

When constructing the diverse antibody libraries of the presentinvention, modified amino acid residues, for example, residues outsidethe traditional 20 amino acids used in most polypeptides, e.g.,homocysteine, can be incorporated into the antibody sequences, such asCDRs, as desired. This can be carried out using art recognizedtechniques which typically introduce stop codons into the polynucleotidewhere the modified amino acid residue is desired. The technique thenprovides a modified tRNA linked to the modified amino acid to beincorporated (a so-called suppressor tRNA of, e.g., the stop codonamber, opal, or ochre) into the polypeptide (see, e.g., Köhrer et al.,PNAS, 98, 14310-14315 (2001)).

In a preferred embodiment, one or more of the above steps arecomputer-assisted. In a particular embodiment, the computer assistedstep comprises, e.g., mining the Kabat database and, optionally,cross-referencing the results against the Vbase sequence directory(Tomlinson, I M. et al., VBASE Sequence Directory. Cambridge, U.K.: MRCCentre for Protein Engineering; 1995). The methods of the presentinvention are amendable to a high throughput approach comprisingsoftware (e.g., computer-readable instructions) and hardware (e.g.,computers, robotics, and chips) for carrying out the various steps.

The oligonucleotides for generation of the libraries herein can besynthesized by known methods for DNA synthesis. Known synthesis methodsinclude the phosphoramidite chemistry (Beaucage and Caruthers,Tetrahedron Letts., 22(20):1859 1862 (1981)), which permits effectiveoligo preparation, especially in the most common 40 80 bp size range,using an automated synthesizer, as described, for example, inNeedham-VanDevanter et al. Nucleic Acids Res., 12:6159 6168 (1984)). Inaddition, oligonucleotides can be synthesized by the triester,phosphite, and H-phosphonate methods, all well known in the art. For areview of the oligonucleotide synthesis methods see, for example,“Oligonucleotide Synthesis: A Practical Approach”, ed. M. J. Gait, JRLPress, New York, N.Y. (1990). Oligonucleotides can also be customordered from a variety of commercial sources, such as, for example, TheMidland Certified Reagent Company (Midland, Tex.), The Great AmericanGene Company (Salt Lake City, Utah), ExpressGen Inc. (Chicago, Ill.),Operon Technologies Inc. (Alameda, Calif.).

If the library is an antibody library, in the next step diversity iscloned into frameworks to produce a diverse antibody library.

The framework scaffold can be selected by methods well known in the art.Thus, the most frequently used frameworks in the database can be chosenfor use as a scaffold, and diversity is cloned into the germlineframeworks. For framework sequence selection, a subset of all availableframework scaffolds determined to have been expressed in response to aparticular antigen are arrayed. By determining the frameworks that aremost frequently expressed in nature in response to a given antigen classan appropriate framework acceptor is selected. For example, to determinethe preferred acceptor frameworks expressed in response to protein-basedantigens, the Kabat database is searched for “protein-directed”frameworks. If preferred acceptor sequences are needed for presentingCDRs against a different antigen class, and/or, acceptor sequences of aparticular species, the Kabat protein sequence filter is setaccordingly. Thus, to determine sequences for use as human therapeuticsagainst protein-based targets, the filter is set to focus only on humanantibody sequences that recognize protein/peptide antigens. This greatlyreduces redundancy in the dataset and sequence information that wouldbias results. Such analysis can be performed for V_(H), V_(κ) and/orV_(λ) genes in a similar manner.

The diverse collections can be incorporated on an acceptor that istarget specific to generate variant collections for antibodyengineering.

The CDR diversity generated can be incorporated into framework regionsby methods known in the art, such as polymerase chain reaction (PCR).For example, the oligonucleotides can be used as primers for extension.In this approach, oligonucleotides encoding the mutagenic cassettescorresponding to the defined region (or portion thereof), such as a CDR,are complementary to each other, at least in part, and can be extendedto form a large gene cassette (e.g., a scFv) using a polymerase, e.g., aTaq polymerase.

In another approach, partially overlapping oligonucleotides aredesigned. The internal oligonucleotides are annealed to theircomplementary strand to yield a double-stranded DNA molecule withsingle-stranded extensions useful for further annealing. The annealedpairs can then be mixed together, extended, and ligated to formfull-length double-stranded molecules using PCR. Convenient restrictionsites can be designed near the ends of the synthetic gene for cloninginto a suitable vector. In this approach, degenerate nucleotides canalso be directly incorporated in place of one of the oligonucleotides.The complementary strand is synthesized during the primer extensionreaction from a partially complementary oligonucleotide from the otherstrand by enzymatic extension with the aid of a polymerase.Incorporation of the degenerate polynucleotides at the stage ofsynthesis simplifies cloning, for example, where more than one domain ordefined region of a gene is mutagenized or engineered to have diversity.

Regardless of the method used, after conversion into double strandedform, the oligonucleotides can be ligated into a suitable expressionvector by standard techniques. By means of an appropriate vector, suchas a suitable plasmid, the genes can be introduced into a cell-freeextract, or prokaryotic cell or eukaryotic cell suitable for expressionof the antibodies.

In a different approach, the desired coding sequence can be cloned intoa phage vector or a vector with a filamentous phage origin ofreplication that allows propagation of single-stranded molecules withthe use of a helper phage. The single-stranded template can be annealedwith a set of degenerate oligonucleotides representing the desiredmutations, elongated and ligated, thus incorporating each analog strandinto a population of molecules that can be introduced into anappropriate host (see, e.g., Sayers, J. R. et al., Nucleic Acids Res.16: 791-802 (1988)).

Various phagemid cloning systems suitable for producing the libraries,such as synthetic human antibody libraries, herein are known in the art,and have been described, for example, by Kang et al., Proc. Natl. Acad.Sci., USA, 88:4363 4366 (1991); Barbas et al., Proc. Natl. Acad. Sci.USA, 88:7978 7982 (1991); Zebedee et al., Proc. Natl. Acad. Sci., USA,89:3175 3179 (1992); Kang et al., Proc. Natl. Acad. Sci., USA, 88:1112011123 (1991); Barbas et al., Proc. Natl. Acad. Sci., USA, 89:4457 4461(1992); and Gram et al., Proc. Natl. Acad. Sci., USA, 89:3576 3580(1992).

The size of the library will vary depending upon the CDR length and theamount of CDR diversity which needs to be represented. Preferably, thelibrary will be designed to contain less than 10¹⁵, 10¹⁴, 10¹³, 10¹²,10¹¹, 10¹⁰, 10⁹, 10⁸, 10⁷, and more preferably, 10⁶ or less antibodiesor antibody fragments.

The libraries constructed in accordance with the present invention maybe also attached to a solid support, such as a microchip, and preferablyarrayed, using art recognized techniques.

The libraries constructed in accordance with the present invention canbe expressed using any methods known in the art, including, withoutlimitation, bacterial expression systems, mammalian expression systems,and in vitro ribosomal display systems.

In a preferred embodiment, the present invention encompasses the use ofphage vectors to express the diverse libraries herein. The methodgenerally involves the use of a filamentous phage (phagemid) surfaceexpression vector system for cloning and expression. See, e.g., Kang etal., Proc. Natl. Acad. Sci., USA, 88:4363-4366 (1991); Barbas et al.,Proc. Natl. Acad. Sci., USA, 88:7978-7982 (1991); Zebedee et al., Proc.Natl. Acad. Sci., USA, 89:3175-3179 (1992); Kang et al., Proc. Natl.Acad. Sci., USA, 88:11120-11123 (1991); Barbas et al., Proc. Natl. Acad.Sci., USA, 89:4457-4461 (1992); Gram et al., Proc. Natl. Acad. Sci.,USA, 89:3576-3580 (1992); Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); and U.S. Pat. Nos. 5,698,426; 5,233,409; 5,580,717; 5,427,908;5,750,753; 5,821,047; 5,403,484; 5,571,698; 5,516,637; 5,780,225;5,658,727; 5,733,743; 5,837,500; 5,969,108; 6,326,155; 5,885,793;6,521,404; 6,492,160, 6,492,123; 6,489,123; 6,342,588; 6,291,650;6,225,447; 6,180,336; 6,172,197; 6,140,471; 5,994,519; 6,969,108;5,871,907; and 5,858,657.

The vector is used to transform a recombinant host cell, which iscultured to allow the introduced phage genes and display protein genesto be expressed, and for phage particles to be assembled and shed fromthe host cell. The shed phage particles are then harvested (collected)from the host cell culture media and screened for desirable antibodybinding properties. Typically, the harvested particles are “panned” forbinding with a preselected antigen. The strongly binding particles arecollected, and individual species of particles are clonally isolated andfurther screened for binding to the antigen. Phages which produce abinding site of desired antigen binding specificity are selected.

It is emphasized that the methods of the present invention are notlimited by any particular technology used for the expression and displayof antibody libraries. Other display techniques, such as ribosome ormRNA display (Mattheakis et al., Proc. Natl. Acad. Sci. USA 91:9022-9026(1994); Hanes and Pluckthun, Proc. Natl. Acad. Sci. USA 94:4937-4942(1997)), microbial cell display, such as bacterial display (Georgiou etal., Nature Biotech. 15:29-34 (1997)), or yeast cell display (Kieke etal., Protein Eng. 10:1303-1310 (1997)), display on mammalian cells,spore display, viral display, such as retroviral display (Urban et al.,Nucleic Acids Res. 33:e35 (2005), display based on protein-DNA linkage(Odegrip et al., Proc. Acad. Natl. Sci. USA 101:2806-2810 (2004);Reiersen et al., Nucleic Acids Res. 33:e10 (2005)), and microbeaddisplay (Sepp et al., FEBS Lett. 532:455-458 (2002)) are also suitable.

In ribosome display, the antibody and the encoding mRNA are linked bythe ribosome, which at the end of translating the mRNA is made to stopwithout releasing the polypeptide. Selection is based on the ternarycomplex as a whole.

In a mRNA display library, a covalent bond between an antibody and theencoding mRNA is established via puromycin, used as an adaptor molecule(Wilson et al., Proc. Natl. Acad. Sci. USA 98:3750-3755 (2001)). For useof this technique to display antibodies, see, e.g., Lipovsek andPluckthun, J. Immunol. Methods. 290:51-67 (2004).

Microbial cell display techniques include surface display on a yeast,such as Saccharomyces cerevisiae (Boder and Wittrup, Nat. Biotechnol,15:553-557 (1997)). Thus, for example, antibodies can be displayed onthe surface of S. cerevisiae via fusion to the α-agglutinin yeastadhesion receptor, which is located on the yeast cell wall. This methodprovides the possibility of selecting repertoires by flow cytometry. Bystaining the cells by fluorescently labeled antigen and an anti-epitopetag reagent, the yeast cells can be sorted according to the level ofantigen binding and antibody expression on the cell surface. Yeastdisplay platforms can also be combined with phage (see, e.g., Van denBeucken et al., FEBS Lett. 546:288-294 (2003)).

For a review of techniques for selecting and screening antibodylibraries see, e.g., Hoogenboom, Nature Biotechnol. 23(9):1105-1116(2005).

The invention will be illustrated by the following, non-limitingExamples.

EXAMPLES

Techniques for performing the methods of the present invention are wellknown in the art and described in standard laboratory textbooks,including, for example, Ausubel et al., Current Protocols of MolecularBiology, John Wiley and Sons (1997); Molecular Cloning: A LaboratoryManual, Third Edition, J. Sambrook and D. W. Russell, eds., Cold SpringHarbor, N.Y., USA, Cold Spring Harbor Laboratory Press, 2001; O'Brian etal., Antibody Phage Display, Methods and Protocols, Humana Press, 2001;Phage Display: A Laboratory Manual, C. F. Barbas III et al. eds., ColdSpring Harbor, N.Y., USA, Cold Spring Harbor Laboratory Press, 2001; andAntibodies, G. Subramanian, ed., Kluwer Academic, 2004. Mutagenesis can,fore example, be performed using site-directed mutagenesis (Kunkel etal., Proc. Natl. Acad. Sci USA 82:488-492 (1985)); DNA Cloning, Vols. 1and 2, (D. N. Glover, Ed. 1985); Oligonucleotide Synthesis (M. J. Gait,Ed. 1984); PCR Handbook Current Protocols in Nucleic Acid Chemistry,Beaucage, Ed. John Wiley & Sons (1999) (Editor); Oxford Handbook ofNucleic Acid Structure, Neidle, Ed., Oxford Univ Press (1999); PCRProtocols: A Guide to Methods and Applications, Innis et al., AcademicPress (1990); PCR Essential Techniques: Essential Techniques, Burke,Ed., John Wiley & Son Ltd (1996); The PCR Technique: RT-PCR, Siebert,Ed., Eaton Pub. Co. (1998); Antibody Engineering Protocols (Methods inMolecular Biology), 510, Paul, S., Humana Pr (1996); AntibodyEngineering: A Practical Approach (Practical Approach Series, 169),McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlowet al., C.S.H.L. Press, Pub. (1999); Large-Scale Mammalian Cell CultureTechnology, Lubiniecki, A., Ed., Marcel Dekker, Pub., (1990). Border etal., Yeast surface display for screening combinatorial polypeptidelibraries, Nature Biotechnology, 15(6):553-7 (1997); Border et al.,Yeast surface display for directed evolution of protein expression,affinity, and stability, Methods Enzymol., 328:430-44 (2000); ribosomedisplay as described by Pluckthun et al. in U.S. Pat. No. 6,348,315, andProfusion™ as described by Szostak et al. in U.S. Pat. Nos. 6,258,558;6,261,804; and 6,214,553; and bacterial periplasmic expression asdescribed in US20040058403A1.

Further details regarding antibody sequence analysis using Kabatconventions may be found, e.g., in Johnson et al., The Kabat databaseand a bioinformatics example, Methods Mol Biol. 2004; 248:11-25; Johnsonet al., Preferred CDRH3 lengths for antibodies with definedspecificities, hit Immunol. 1998, December; 10(12):1801-5; Johnson etal., SEQHUNT. A program to screen aligned nucleotide and amino acidsequences, Methods Mol Biol. 1995; 51:1-15. and Wu et al., Lengthdistribution of CDRH3 in antibodies; and Johnson et al., Proteins. 1993May; 16(1):1-7. Review).

Further details regarding antibody sequence analysis using Chothiaconventions may be found, e.g., in Chothia et al., Structuraldeterminants in the sequences of immunoglobulin variable domain, J MolBiol. 1998 May 1; 278(2):457-79; Morea et al., Antibody structure,prediction and redesign, Biophys Chem. 1997 October; 68(1-3):9-16; Moreaet al., Conformations of the third hypervariable region in the VH domainof immunoglobulins; J Mol Biol. 1998 Jan. 16; 275(2):269-94; Al-Lazikaniet al., Standard conformations for the canonical structures ofimmunoglobulins, J Mol Biol. 1997 Nov. 7; 273(4):927-48. Bane et al.,Structural conservation of hypervariable regions in immunoglobulinsevolution, Nat Struct Biol. 1994 December; 1(12):915-20; Chothia et al.,Structural repertoire of the human VH segments, J Mol Biol. 1992 Oct. 5;227(3):799-817 Conformations of immunoglobulin hypervariable regions,Nature. 1989 Dec. 21-28; 342(6252):877-83; and Chothia et al., ReviewCanonical structures for the hypervariable regions of immunoglobulins, JMol Biol. 1987 Aug. 20; 196(4):901-17).

Further details regarding Chothia analysis are described, for example,in Morea V, Tramontano A, Rustici M, Chothia C, Lesk A M. Conformationsof the third hypervariable region in the VH domain of immunoglobulins. JMol Biol. 1998 Jan. 16; 275(2):269-94; Chothia C, Lesk A M, Gherardi E,Tomlinson I M, Walter G, Marks J D, Llewelyn M B, Winter G. Structuralrepertoire of the human VH segments. J Mol Biol. 1992 Oct. 5;227(3):799-817; Chothia C, Lesk A M, Tramontano A, Levitt M, Smith-GillS J, Air G, Sheriff S, Padlan E A, Davies D, Tulip W R, et al.Conformations of immunoglobulin hypervariable regions. Nature. 1989 Dec.21-28; 342(6252):877-83; Chothia C, Lesk A M. Canonical structures forthe hypervariable regions of immunoglobulins. J Mol Biol. 1987 Aug. 20;196(4):901-17; and Chothia C, Lesk A M. The evolution of proteinstructures. Cold Spring Harb Symp Quant Biol. 1987; 52:399-405.

Further details regarding CDR contact considerations are described, forexample, in MacCallum R M, Martin A C, Thornton J M. Antibody-antigeninteractions: contact analysis and binding site Topography. J Mol Biol.1996 Oct. 11; 262(5):732-45.

Further details regarding the antibody sequences and databases referredto herein are found, e.g., in Tomlinson I M, Walter G, Marks J D,Llewelyn M B, Winter G. The repertoire of human germline VH sequencesreveals about fifty groups of VH segments with different hypervariableloops. J Mol Biol. 1992 Oct. 5; 227(3):776-98; Li W, Jaroszewski L,Godzik A. Clustering of highly homologous sequences to reduce the sizeof large protein databases. Bioinformatics. 2001 March; 17(3):282-3;[VBDB] www.mrc-cpe.cam.ac.ukkbase-ok.php?menu=901; [KBTDB] database.com;[BLST] www.ncbi.nlm.nih.gov/BLAST/[CDHIT]bioinformatics.ljcrfedu/cd-hi/; [EMBOSS]www.hgmp.mrc.ac.uk/Software/EMBOSS/; [PHYLIP]evolution.genetics.washington.edu/phylip.html; and [FASTA]fasta.bioch.virginia.edu.

Example 1

Frequency Analysis of Antibody Light Chain V_(κ)1 CDR1, 2, and 3Sequences

In a first step, 2374 human antibody V_(κ)1 light chain variable domainsequences were collected from the Kabat Database of Sequences ofProteins of Immunological Interest. For each sequence, the gene sequencewas translated into the corresponding amino acid sequence, and the aminoacid sequences were positionally aligned, following the Kabat numberingsystem.

Next, the collection of V_(κ)1 light chain sequences obtained wasfiltered by selecting sequences having amino acids “RV” at positions and18-19, and applying the following length restrictions: CDR1=7 aminoacids, CDR2=10 amino acids, and CDR3=8 amino acids. By applying thesefilters, the originally 2374-member collection was reduced to 771members.

By using only entries containing complete unambiguous sequences from the“RV” motif preceding DR1 through a complete CDR3 sequence, the number ofV_(κ)1 light chain variable domain sequences was further reduced to 383.

Subsequently, the sequences were aligned, the occurrence amino acids ateach position was tabulated, and the distribution of the 20 naturallyoccurring amino acids at each position was calculated to produce thepositional frequency-based database of CDR domain diversity based onabsolute usage of amino acids by position. The results of thistabulation are shown in FIG. 2.

The datasets set forth in FIG. 2 were further filtered by reporting onlyamino acid usage that was above 10% for any given position. The resultsare set forth in FIG. 3. In order to assess the effect of the percentageusage specified on diversity, another dataset was created by includingonly amino acid usage that was above 5%. The results are shown in FIG.4. From comparing the datasets of FIGS. 3 and 4, it is clear thatgreater coverage of diversity is achieved by requiring a lowerpercentage of amino acid usage.

As shown in FIG. 5, in order to encode the light chain CDR1 diversityset forth in FIG. 4, 128 combinatorial oligonucleotides or 16 degeneratecombinatorial oligonucleotides need to be synthesized. The bases neednot be equimolar and can be tuned to bias amino acid usage to reflectfrequencies found in the present analysis, and even include residues notincluded in the frequency tables. Alternatively or in addition, residuesincluded in the frequency tables may be omitted, for example to furtherreduce the number of oligonucleotides needed for synthesis.

Example 2 Design of V_(H)3 Heavy Chain Synthetic Library Diversity

Analyzing V_(H)3 heavy chain polypeptide sequences, 10 amino acids inlength, obtained from the Kabat database of antibody sequencesessentially as described in Example 1, the data shown in FIG. 6 wasgenerated. As shown in FIG. 6, a CDR3 diversity of 3.3×10⁵ representingat least 75% positional coverage for all positions except residue 97 canbe provided by using only 96 degenerate oligonucleotides, settingdifferent threshold percent usages for various amino acid positions.Thus the threshold percentage usage was 10% for positions 93, 94, 100and 101; 5% for positions 95, 96, 98, and 99; 4% for position 97; and 3%for position 100A. The oligonucleotide sequences needed to synthesizethis diversity are shown in FIG. 7.

Example 3 Making a Semi-Synthetic Antibody Library

As previously described, the analysis and generation of the V_(H) CDRdiversity can be tuned to contextually reflect compositions forproductive and specific pairings with κ or λ light chains, (i.e.pairings resulting in antibodies specifically binding a target antigen).These synthetic V_(H) repertoires need not exclusively be paired withsynthetic light chain repertoires, but can be combinatorially clonedwith collections of lymphocyte derived light chains. In practice, acollection of κ and λ light chains is separately cloned into a phagedisplay vector followed by cloning of either the individual heavy chainvariable region frameworks for subsequent introduction of diversity or acollection of pre-diversified variable region frameworks. In eithercase, the light chain compatibly paired heavy chain variable regions areexpected to more productively pair with the corresponding light chains.

Example 4 Engineering Improved Antibodies by Creating Libraries ofVariants Upon a Base Clone

In a matter analogous to introducing productive diversity upon agermline acceptor frameworks for creating de novo immunoglobulinrepertoires, target specific mutagenesis libraries are created for aspecific antibody or defined collection of antibodies. Such librariesare useful in the task of antibody engineering especially in the fieldof affinity maturation. Starting with a monoclonal antibody of interest,the defining characteristics are determined, which are captured in thepreviously defined diversity influencing elements of the presentinvention, such as, germline framework origin, light chain type, andlight chain and heavy chain CDR lengths. After determining these orsimilar characteristics the next step is to refer to database sequencesthat correspond these parameters. Subsequent to identifying thecorresponding sets of sequences an analysis, similar to that describedearlier, is performed to examine subset repertoire diversity and thengenerate the corresponding multi-degenerate oligonucleotides necessaryto encode the desired diversity. These multi-degenerate oligonucleotidesare then cloned as single or combinatorial CDR collections. As it ismore likely to find synergistic improvements with multi-CDR mutagenesisthe creation of combinatorial CDR mutagenesis libraries is preferred.Using the multi-degenerate oligonucleotides from the analysis describedabove rationally creates and re-diversifies antibodies according topositional diversity with respect to human bias and preference. It isimportant to note that in instances where any of the light chain CDRsequences or heavy chain CDR1 or CDR2 sequences diverge from thegermline sequence the corresponding germline-encoding oligonucleotide isalso included into the combinatorial CDR library. This inclusion ofgermline encoding oligonucleotides allows for backcrossing of germlinesequences to create more productive CDR combinations.

This “diversity reincorporation scheme” is also useful in engineering are-diversified set of antibodies from an existing synthetic antibodyclone. As the potential diversity of the synthetic libraries created inaccordance with the present invention exceeds the limits of currentlyavailable techniques to display and select all members, it is verylikely that any discovered target specific clones represent only afraction of the possible solutions present and accessible at theDNA-level of any of the typically screened libraries. Thus, afteridentifying in a library of the present invention, following four roundsof panning, an anti-EGF antibody, the originally designed diversity wascombinatorially reintroduced into the clone to create a new set ofvariants. These new sets of variants were then re-screened by panning onEGF and in each successive round the stringency of binding and washingwas increased. The net result created pools of EGF binding phage thatenriched to greater levels over background than those found in theoriginal panning.

Example 5 Design of a Cytokine Theme Library

In order to create productive libraries for the discovery of newanti-cytokine antibodies, a productive anti-TNF-α antibody, HUMIRA®(adalimumab) was selected as a basic theme. HUMIRA® (adalimumab) is arecombinant human IgG1 monoclonal antibody created using phage displaytechnology resulting in an antibody with human-derived heavy and lightchain variable regions and human IgG1:κ constant regions.

To determine the germline origin of the heavy chain of parental antibodyD2E7, the framework region was analyzed. This was accomplished bymasking the CDRs of D2E7 and of the human germline VH genes. Next theremaining sequences between FR1 and FR3 for D2E7 was aligned by BLASTalgorithm against all of the human germline VH genes. As shown in FIG.8, the D2H7 V_(H) region showed greatest similarity to V_(H)3_3-09. Thedendogram alignment set forth in FIG. 9 shows the same result. In asimilar fashion the light chain of parental antibody D2E7 found to bemost similar to V_(κ)1 A20 (FIGS. 10 and 11).

The frequency analysis of antibody light chain V_(κ)1 CDR1, CDR2, andCDR3 sequences described in Example 1 was modified by setting thethreshold percent usage filter to 6%. As shown in FIG. 12, with thisfilter the sum usage for all amino acid positions, except position 91,is over 80%, which accommodates a library diversity of 9×10⁶, and thisdiversity can be generated by 30 degenerate oligonucleotides.

Next, 5971 human antibody heavy chain variable domain sequences werecollected from the Kabat Database of Sequences of Proteins ofImmunological Interest. For each sequence, the gene sequence wastranslated into the corresponding amino acid sequence, and the aminoacid sequences were positionally aligned, following the Kabat numberingsystem.

The heavy chain variable domain collection was then subjected to thefollowing filters:

1. V_(H)3 sequences containing “CAAS” at amino acid positions 22-25(1530 of 5971 members);

2. sequences combined with κ light chains, CDR1=6 amino acids andCDR2=13 amino acids (226 of 1530 members)

3. Including only members containing complete sequences from “CAAS”preceding CDR1 through a complete CDR2 sequence (180 of 226 members).

Subsequently, the sequences were aligned, the occurrence amino acids ateach position was tabulated, and the distribution of the 20 naturallyoccurring amino acids at each position was calculated to produce thepositional frequency-based database of CDR domain diversity based onabsolute usage of amino acids by position. The results of thistabulation are shown in FIG. 13.

The datasets set forth in FIG. 13 were further filtered by reportingonly amino acid usage that was at least 10% for any given position. Asshown in FIG. 14, using this filter, in CDR2 the sum amino acid coverageat positions 52, 52A, 55, and 58 is less than 75%. To accommodate agreater coverage, the required percent usage has been reduced from 10%to 5%. As shown in FIG. 15, this change has resulted in raising the sumamino acid usage for all positions to greater than 75%.

Applying the 5% usage filter both CDR1 and CDR2 4 degenerateoligonucleotides required for the synthesis of the CDR1 regions, CDR2diversity can be encoded by 28 degenerate oligonucleotides (see FIG.16). Thus, using a total of 28 degenerate oligonucleotides, a totaldiversity of 1.5×10⁸ can be achieved, providing more than 80% positionalcoverage.

In the next step, from the 5971 human antibody heavy chain variabledomain sequences described above, sequences V_(H)3 sequences 13 aminoacids in length were compiled, regardless of isotype. The requiredpercent amino acid usage for each position was set to 4%, except atamino acid positions 93, 94 and 101, where the threshold was set to 4%usage. The results are set forth in FIG. 17. By setting thesethresholds, a synthetic V_(H)3 heavy chain synthetic library with atotal diversity of 7.5×10⁹ can be prepared by using 384 degenerateoligonucleotides. As shown, residues in the CD3 region shows a goodagreement with the corresponding residues in the parent antibody D2E7.

Example 6 Design of a Hapten Themed Antibody Library

The objective of this method is to design productive libraries for theidentification of new anti-hapten antibodies.

The design started with an anti-digoxigenin (anti-DIG) antibody (Dorsam,H. et al., FEBS Lett. 414:7-13 (1997)). The Ig λ light chain variableregion sequence (SEQ ID NO: 1) and the heavy chain variable regionsequence (SEQ ID NO: 2) for this antibody are shown in FIG. 18.

In order to determine the germline origin of the heavy and light chainsof this parental antibody were analyzed. As shown in FIG. 19, V_(L)-1gis most similar to the light chain, and V_(H)3-23 is most similar to theheavy chain, therefore, the CDRs were put in this environment in orderto create a productive library for identification of anti-haptenantibodies.

Next, the light chain CDR1 and CDR2 sequences were analyzed as describedin the previous examples for λ length matched V_(L) framework residues.The required percent amino acid usage for each position was set to 6%,so that no individual sequences were reported below 6%. As shown in FIG.20, this filter provide an excellent coverage for each amino acidposition. Performing a similar analysis for H3 length matched (8 aminoacids) heavy chain, but applying a 6.25% filter, the sum amino acidcoverage, including all positions, was above 75% (FIG. 21).

Example 7 Cytokine (IFN-α) Analysis and Library Creation

IFN is a generic term for cytokines having anti-viral activity, amongwhich those produced from leukocytes or lymphoblastic cells bystimulation with virus or double stranded nucleic acids are termed asIFN-α. IFN-α has a variety of activities including anti-viral activityand cellular growth-suppressing activity, which activities have beenfound to be useful in the treatment of a variety of diseases such ashepatitis type B and type C infections, and cancer. Analysis ofsequences of IFN-α genes cloned from a variety of DNA libraries hasrevealed that IFN-α exists in several subtypes. For example, for theIFN-α2 gene, three additional types (α2a, .α2b, and .α2c) have beenidentified. Altogether, there are over 20 currently known IFN-αsubtypes. Additional known subtypes include, for example, IFN-α1a,IFN-α1b, IFN-α4a, IFN-α4b, IFN-α5, IFN-α6, etc. It has been demonstratedthat many of the IFN-α subtypes differ in their biological activitiesand other biological properties. Therefore, libraries created based uponthe existing natural diversity among members of the IFN-α family findutility in generating IFN-α polypeptides with new and improvedproperties, such as increased potency, decreased immunogenicity,increased half-life, improved proteolytic stability.

As a first step for creating a diverse IFN-α library, eleven 189 aminoacids long gene products were identified. Amino acid residues 32-38 ofthese IFN-α polypeptides were aligned with each other and the residuefrequency usage was determined, as shown in FIG. 22. When the thresholdpercent amino acid usage is set to 9%, 100% coverage can be achievedusing 2 degenerate oligonucleotides (see, FIGS. 22 and 23). As shown inFIG. 23, with a non-degenerate design 40 oligonucleotides are needed toprovide the required coverage.

Once the library is prepared, screening for desired novel properties canbe performed by methods known in the art. Thus, increased potency can betested in standard biological assays, such as by biopanning aphage-displayed IFN-α library. members with increased half-life can beidentified, for example, by biopanning a phage-displayed library againstan IFN-α receptor, or by exposing members of the library to one or moreserum proteases. Decreased immunogenicity can be tested, for example, byidentifying the peptides or polypeptides present in the library thatshow the least binding to MHC molecules, or by testing T cell epitopepresentation of whole proteins directly.

These and numerous additional tests are well known to those of ordinaryskill in the pertinent art.

Example 8 Chemically Probed Antibody Collections

The present example shows the creation of CDR3 heavy chain diversityusing probe sets designed based on chemical principles.

Amino acids can be divided into seven groups, characterized by small,nucleophilic, hydrophobic, aromatic, acidic, amide, and basic side-chainchemical functionalities, respectively (FIG. 24). The top left panel inFIG. 25 shows the one-letter symbols of amino acids present in each ofthe seven groups. Nine amino acids (A, S, H, L, P, Y, D, Q, R),representative of the different side-chain chemistries, were selected.As shown in the rest of FIG. 25, the highlighted nine amino acids can beencoded, and thus the side-chain chemistry diversity can be captured, bynine codons or 2 degenerate codons. (B=C, G, or T; M=A or C; Y=C or T.D=A, G, or T.)

The native heavy chain CDR3 sequence contains a high degree of chemicaldiversity (around 60% or more). It has been determined that a similarchemical diversity can be generated, by combinatorial denegerateoligonucleotide synthesis, using 128 degenerate oligonucleotides. Thedesign of the corresponding degenerate oligonucleotides is shown in FIG.27. As set forth in FIG. 26, this approach covers a majority of thenaturally occurring diversity and provides broad interactivechemistries.

This chemically probed diversity approach can be used on its own, or incombinations with any of the other methods of the present invention, inorder to produce combinatorial libraries with desired properties.

Although in the foregoing description the invention is illustrated withreference to certain embodiments, it is not so limited. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.Thus, while the invention is illustrated with reference to antibodylibraries, it extends generally to all peptide and polypeptidelibraries.

All references cited throughout the specification are hereby expresslyincorporated by reference.

1. A method for diversity analysis of a database comprising relatedamino acid sequences characterized by at least one shared sequencemotif, comprising the steps of: (a) aligning said related amino acidsequences; (b) creating a first dataset by applying a predeterminedcombination of two or more filters to said related amino acid sequencescomprising said shared sequence motif; (c) analyzing said first datasetfor positional amino acid usage frequency within said shared sequencemotif; and (d) creating a second dataset characterized by a minimumthreshold amino acid usage frequency at one or more amino acid positionswithin said shared sequence motif.
 2. The method of claim 1 wherein saidrelated amino acid sequences comprise antibody heavy or light chainsequences.
 3. The method of claim 2 wherein said shared sequence motifis a CDR sequence selected from the group consisting of CDR1, CDR2 andCDR3 sequences.
 4. The method of claim 3 wherein in step (b) saidpredetermined combination of filters is selected from the groupconsisting of (1) the isotype of said antibody heavy or light chain; (2)the length of one or more of said CDR1, CDR2 and CDR3 sequences; (3) thepresence of one or more predetermined amino acid residues at one or morepredetermined positions within one or more of said CDR1, CDR2 and CDR3sequences; (4) type of framework; (5) antigen to which said antibodybinds; (6) affinity of said antibody; and (7) positional amino acidresidues outside said CDR sequences.
 5. The method of claim 6 wherein atleast one of the antibody heavy and/or light chain CDR1, CDR2 and CDR3sequences is size matched.
 6. The method of claim 5 wherein anadditional filter is the isotype of said antibody heavy and/or lightchain sequences.
 7. The method of claim 3 wherein said positional aminoacid usage frequency is between at least about 3% and at least about15%.
 8. The method of claim 3 wherein the same positional amino acidusage frequency characterizes each amino acid within said CDR sequence.9. The method of claim 3 wherein the positional amino acid usagefrequencies differ at at least two amino acid residues within said CDRsequence.
 10. The method of claim 4 wherein said predeterminedcombination of filters includes the type of framework.
 11. The method ofclaim 2 wherein both antibody heavy and light chain sequences areanalyzed, and the antibody heavy chain sequences are paired topredetermined antibody light chain characteristics, or the antibodylight chain sequences are paired to predetermined antibody heavy chaincharacteristics.
 12. The method of claim 2 wherein said related antibodysequences are from at least one functional antibody.
 13. The method ofclaim 12 wherein one of said filters applied in step (b) is the germlinesequence most similar to the framework sequence of the heavy and/orlight chain of said functional antibody.
 14. The method of claim 12wherein said functional antibody binds to a polypeptide selected fromthe group consisting of cell surface and soluble receptors, cytokines,growth factors, enzymes; proteases; and hormones.
 15. The method ofclaim 2 wherein in step (d) a minimum threshold amino acid usagefrequency is assigned to at least the majority of amino acid positionswithin said shared sequence motif.
 16. The method of claim 2 whereinsaid minimum threshold amino acid usage frequency is set to provide aminimum sum amino acid usage for the majority of amino acid positionswithin said shared sequence motif.
 17. The method of claim 16 whereinsaid minimum threshold amino acid usage frequency is set to provide aminimum sum amino acid usage for all amino acid positions within saidshared sequence motif.
 18. The method of claim 17 wherein said minimumsum amino acid usage is between at least about 60% and at least about90%.19. The method of claim 2 further comprising the step ofsynthesizing a physical library of related amino acid sequences that isdesigned with the aid of the datasets identified.
 19. A method of makinga library of antibody heavy or light chains, comprising the steps of (a)aligning a plurality of antibody heavy or light chain sequencescomprising one or more CDR sequence motifs; (b) creating a first datasetby applying a predetermined combination of two or more filters to saidantibody heavy or light chain sequences; (c) analyzing said firstdataset for positional amino acid usage frequency within at least one ofsaid CDR sequence motifs; (d) creating a second dataset characterized bya minimum threshold amino acid usage frequency at one or more amino acidpositions within said CDR sequence motif; and (e) synthesizing anantibody heavy or light chain library designed with the aid of the firstand second datasets identified.
 20. The method of claim 20 wherein saidlibrary is synthesized by generating a discrete number of defined ordegenerate oligonucleotides such that only defined amino acids aregenerated.
 21. The method of claim 20 wherein the diversity of thephysical library produced exceeds the diversity of a library which is aphysical representation of the datasets identified.
 22. The method ofclaim 22 wherein at least one amino acid not meeting the minimumthreshold amino acid usage frequency is also synthesized to provide saiddiversity.
 23. The method of claim 20 wherein the diversity of thephysical library produced is less than the diversity of a library whichis a physical representation of the datasets identified.
 24. The methodof claim 24 wherein not all amino acids meeting the minimum thresholdamino acid usage frequency are synthesized.
 25. The method of claim 20wherein said CDRs are cloned into a scaffold of framework sequences. 26.The method of claim 26 wherein said framework sequences are the mostfrequently used framework sequences in the database comprising saidCDRs.
 27. The method of claim 20 wherein said physical library isexpressed using a prokaryotic or eukaryotic expression system.
 28. Themethod of claim 20 wherein said physical library is expressed anddisplayed using a phagemid display, mRNA display, microbial celldisplay, mammalian cell display, microbead display technique, antibodyarray, or display based on protein-DNA linkage.
 29. The method of claim20 wherein said library is screened for one or more chemical and/orbiological properties of its members.
 30. The method of claim 30 whereinsaid biological property is selected from the group consisting ofhalf-life, potency, efficacy, binding affinity, and immunogenicity. 31.The method of claim 20 comprising the introduction of amino acidside-chain diversity at one or more amino acid positions.
 32. The methodof claim 32 wherein said amino acid side-chain diversity is introducedby providing amino acid residues with at least two different side-chainchemical functionalities at said amino acid position or positions. 33.The method of claim 33 wherein at least 30% to at least 50% of all aminoacid chemistries are represented at each amino acid position.
 34. Themethod of claim 33 wherein said side-chain diversity is introduced byusing combinatorial degenerate oligonucleotide synthesis.
 35. A methodof producing a combinatorial library of peptide or polypeptidesequences, comprising introducing amino acid side-chain chemicaldiversity into said peptide or polypeptide sequences at two or moreamino acid positions, using combinatorial oligonucleotide synthesis. 36.The method of claim 36 wherein said amino acid side-chain chemicaldiversity is designed to mimic naturally occurring diversity in saidpeptide or polypeptide sequences.
 37. The method of claim 36 whereinsaid library is an antibody library.
 38. The method of claim 38 whereinsaid antibody library comprises antibody heavy chain variable domainsequences.
 39. The method of claim 38 wherein said library comprisesantibody light chain variable domain sequences.
 40. The method of claim38 wherein said library is a combinatorial single-chain variablefragment (scFv) library.
 41. The method of claim 38 wherein saidantibody library is a library of Fab, Fab′, or F(ab′)₂ fragments.